title: The CpG island in the murine foxl2 proximal promoter is differentially methylated in primary and immortalized cells.
abstract: Forkhead box L2 (Foxl2), a member of the forkhead transcription factor family, plays important roles in pituitary follicle-stimulating hormone synthesis and in ovarian maintenance and function. Mutations in the human FOXL2 gene cause eyelid malformations and premature ovarian failure. FOXL2/Foxl2 is expressed in pituitary gonadotrope and thyrotrope cells, the perioptic mesenchyme of the developing eyelid, and ovarian granulosa cells. The mechanisms governing this cell-restricted expression have not been described. We mapped the Foxl2 transcriptional start site in immortalized murine gonadotrope-like cells, LβT2, by 5' rapid amplification of cDNA ends and then PCR amplified approximately 1 kb of 5' flanking sequence from murine genomic DNA. When ligated into a reporter plasmid, the proximal promoter conferred luciferase activity in both homologous (LβT2) and, unexpectedly, heterologous (NIH3T3) cells. In silico analyses identified a CpG island in the proximal promoter and 5' untranslated region, suggesting that Foxl2 transcription might be regulated epigenetically. Indeed, pyrosequencing and quantitative analysis of DNA methylation using real-time PCR revealed Foxl2 proximal promoter hypomethylation in homologous compared to some, though not all, heterologous cell lines. The promoter was also hypomethylated in purified murine gonadotropes. In vitro promoter methylation completely silenced reporter activity in heterologous and homologous cells. Collectively, the data suggest that differential proximal promoter DNA methylation may contribute to cell-specific Foxl2 expression in some cellular contexts. However, gonadotrope-specific expression of the gene cannot be explained by promoter hypomethylation alone.
authors.affiliation: Department of Pharmacology and Therapeutics, McGill University, Montréal, Québec, Canada ; Department of Medicine, University of California Los Angeles, Los Angeles, California, United States of America.
authors.forename: Stella
authors.initials: S
authors.lastname: Tran
authors.forename: Ying
authors.initials: Y
authors.lastname: Wang
authors.forename: Pankaj
authors.initials: P
authors.lastname: Lamba
authors.forename: Xiang
authors.initials: X
authors.lastname: Zhou
authors.forename: Ulrich
authors.initials: U
authors.lastname: Boehm
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Bernard
mesh_terms: D020121:5' Untranslated Regions; D000818:Animals; D001483:Base Sequence; D002461:Cell Line, Transformed; D018899:CpG Islands; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D005260:Female; D000074842:Forkhead Box Protein L2; D051858:Forkhead Transcription Factors / Q000235:genetics* / Q000378:metabolism; D052681:Gonadotrophs / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D009928:Organ Specificity; D011401:Promoter Regions, Genetic; D052684:Thyrotrophs / Q000166:cytology / Q000378:metabolism
chemical_list: D020121:5' Untranslated Regions; D000074842:Forkhead Box Protein L2; D051858:Forkhead Transcription Factors; C498893:Foxl2 protein, mouse